Figures and Table :4-O-carboxymethylascochlorin inhibits expression levels of on inflammation-related cytokines and matrix metalloproteinase-9 through NF‑κB/MAPK/TLR4 signaling pathway in LPS-activated RAW264.7 cells

FIGURE. 1. Chemical structures of 4-O-Carboxymethylascochlorin (AS-6) and ascochlorin (ASC) and effects of AS-6 on viability of RAW 264.7 cells.

(A) 4-O-Carboxymethylascochlorin: AS-6 (Chemical structure). (B) Ascochlorin: ASC (Chemical structure). (C) Cell viability measured by MTT assay. 1 × 104 cell/well cells were treated with 0, 30, or 50 μM of AS-6 and LPS (100 ng/mL) for 24 h.

FIGURE. 2. Effects of AS-6 on PGE2 and NO production in macrophage cells.

(A) 5 X 105 cells/well cells were treated with LPS (100 ng/mL) alone or with AS-6 (0-50 μM) for 24 h. NO in the medium was assessed using Griess assays. (B) 5 X 105 cells/well cells were treated with LPS (100 ng/mL) alone or with AS-6 (0-50 μM) for 24 h. PGE2 in the medium was assessed by ELISA. (C), (D) Inhibition curves of the AS-6-treated NO and PGE2 productions at the different concentrations (0,5,10,20,30,40 and 50 μM). Results shown are representative of three independent experiments. They are presented as mean ± SEM. *p < 0.05 and **p< 0.01, significant differences from LPS treated cells. NT, no treatment.

FIGURE. 3. Effect of AS-6 on protein and expression levels of iNOS, COX-2, and MMP-9 in macrophage cells.

RAW 264.7 cells were treated with 0-50 μM AS-6 and then co-treated with 100 ng/mL LPS for 24 h. (A), (B) Protein and mRNA levels were examined by Western blot and RT-PCR respectively. (C) Zymography results of MMP-9 activity. Results shown are representative of three independent experiments. They are presented as mean ± SEM. *p < 0.05 and **p< 0.01, significant differences from the LPS treated cells. NT, no treatment.

FIGURE. 4. Effects of AS-6 on LPS-induced TNF-α, IL-6 and IL-1β cytokine levels in

macrophage cells.

Macrophage cells were treated with 0-50 μM AS-6 and then co-treated with 100 ng/mL LPS for 24 h. (A), (B), (C) 0-50 μM AS-6 dose-dependently attenuated LPS-induced transcription levels and TNF-α, IL-1β, and IL-6 inflammatory cytokines using ELISA. (D) mRNA levels of cytokines levels of TNF-α, IL-6, and IL-1β were analyzed by RT-PCR. Results shown are representative of three independent experiments. Results are presented was mean ± SEM. *p < 0.05 and **p < 0.01 indicate significant differences from LPS treated cells. NT, no treatment; L, LPS; L+A, LPS + AS-6; A, AS-6.

FIGURE .5. Effects of AS-6 on LPS-stimulated NF-kB nuclear translocation in RAW macrophage cells. (A), (B) RAW cells were pre-treated with AS-6 (30 μM) for 60 minutes before treatment with 100 ng/mL LPS for 0 - 240 minutes time dependently. Cells were harvested and separated into nuclear and cytosolic extracts. Nuclear extracts were subjected to translocation to the nuclear region of NF-κB subunit by Western blot. (C) Transfer of NF-κB to the nucleus was detected by immunofluorescence assay. Macrophage cells were immune-stained by FITC and Hoechst. Scale bars, 5 μm. NE, Nuclear extracts; CE, Cytosolic extract; A, AS-6; L, LPS; L+A, LPS with AS-6; NT, no treatment.

FIGURE .6. Effect of AS-6 on LPS-activated TLR4, MyD-88, and MAPK signaling pathway in RAW 264.7 cells.

RAW cells were pre-treated with AS-6 (50 μM) for 30 minutes before treatment with 100 ng/mL LPS for 60 minutes. (A) Levels of phosphorylated ERK, p38, and JNK were assessed by Western blot analysis. (B) TLR4 and MyD-88 protein levels were determined by Western blot analysis. Levels of ERK, JNK, p38, TLR4, and MyD-88 were indicated by loaded protein as individual control. Results shown are representatives of three independent experiments. Results are presented as mean ± SEM. *p < 0.05 and **p < 0.01 indicate significant differences from LPS treated cells. NT, no treatment.

FIGURE. 7. A schematic illustration showing anti-inflammatory responses in RAW 264.7 cells after treatment with AS-6.

AS-6 inhibited inflammatory response in RAW 264.7 cells through suppression of TLR4, MyD-88, NF-κB, p-ERK1/2, p-JNK, p-p38, iNOS, COX-2, MMP-9, TNF-a, IL-1B, and IL-6.